Screening And Identification Of The Antitumor Strains From Symbiotic Microbial Isolates And Active Compounds Thereof

Cancer is one of the most life-threatening diseases in the world now, and current pharmaceuticals used in treating cancer can only relief symptom, but could not completely cure the cancer. Thus development of antitumor drugs is always a hot aspect in medicine researches.Microorganisms have a role in keeping a dynamic equilibrium in nature mass circulation process, among them varies of microorganisms have kept a symbiotic/epiphytic relationship with plants or animals. Depend on the difference of the hosts’ in vivo environment, those symbiotic microbes usually have shown unique metabolic routes or significant bioactivity. Therefore they have been widely considered as an important source of bioactive natural products,In this thesis, we used human Cervical Carcinoma HeLa cell as a screening model, screened for the antitumor active strains from some symbiotic microbes which were isolated from sponge and plant tissues. In total,252extracts from microbial strain fermentation broth were screened by useing MTT method, SRB method as well as dye exclusion method. Seven strains have decent antitumor activity, and among them an endophytic fungus YX-5showed the remarkable activity. This strain was then identified; the culture condition and the bioactive products stability of this strains were also investigated. After large scale fermentation, the bioactive substances of strain YX-5were isolated and identified by using some chemical approaches such as silicon gel column chromatography and reversed-phrase column chromatography, HPLC, GC-MS and NMR. Through these study, it could provide some practical basis for the development of environmental microbial resource and natural antitumor substances. The detailed results are listed here below:(1) Screening results from SRB method, MTT method and dye exclusion method shown that about28%strains had some antitumor activity against HeLa cells with the inhibition rates above50%at the test concentration of100μg/mL. And the tendencies of screening results from all three methods were approximately the same with good correlation. Among all of the active strains,6bacterial stains and1fungal strain were found exhibiting high and stable antitumor activity, their inhibition rates against HeLa cells were all above80%at100μg/mL, and among them, YX-5showed the highest acitivity with an IC50of5.33μg/mL.(2) By using SEM and colony morphorlogy observation, as well as the ITS rDNA sequencing analysis, the fungus YX-5was primary identified as Aspergillus oryzae. (3) The culture condition of YX-5, including pH, temperature, medium component, rotate speed and culture duration, were studied by measuring the mycelia dried weight, metabolic products yield and antitumor activity. The results showed that the optimum culture condition of YX-5was using GPY medium with pH7.0,110rpm, at25℃for7days.(4) The stability of YX-5bioactive products was also investigated by measuring its antitumor activity after treated with different levels of temperature, pH, UV and protease. It was found that metabolic products of YX-5have a relatively good stability. It can withstand UV and protease treatments to some extend, and only had a decrease activity when exposed to high temperature (above100℃) or in alkaline solutions.(5) YX-5was fermented in large scale (25L), and the cell free culture broth was extracted with ethyl acetate. In total,5.15g of crude extract was obtained. Base on the polarity, the crude extract was primarily separated into three parts, including n-hexane, dichloromethane and65%methanol aqueous phases. Result from SRB method showed that bioactive fraction was mainly existed in the dichloromethane phase. We used TLC to choose solvent system to isolate bioactive dichloromethane phase extract. The results showed that a solvent system of dichloromethane and methanol was more suitable for the sample seperation. The fraction eluted with10%methanol on silica gel column chromatography showed good bioactivity. After futher two ODS reverse phase column, we traced bioactivity of isolated sample and found that subfractions no.10to15have antitumor activity. So we merged these subfractions,and identified bioactive products by using GC-MS and1H-NMR. Bioactive products were aspergillic acids and cyclic dipeptides.